Chondrocytes are mesenchymal cells that have a characteristic phenotype based primarily on the type of extracellular matrix they produce. The precursor cells produce type I collagen but when they become committed to the chondrocyte lineage, they synthesize type II collagen. In addition, committed chondrocytes produce proteoglycan aggregate, called aggrecan, which has glycosaminoglycans that are highly sulfated.
For most cartilaginous tissues, chondrocytes are relatively sparsely distributed in the extracellular matrix; mitosis occurs, but at a low rate; and in part because the tissue is essentially not vascularized, the tissue remains stable over time. A consequence of this is that when damage does occur, it repairs slowly, if at all.
Where cartilage interfaces with bone, the chondrocytes continue to mature along the endochondral pathway. Eventually they hypertrophy, degrade the proteoglycan aggregate, and calcify their matrix. This occurs at different rates, depending on the tissue site, the age of the animal, and the presence of disease or abnormal distribution of force.
The region at the interface of articular cartilage and subchondral bone is called the tidemark. During aging the cartilage becomes thinner as the tidemark continues to form. In osteoarthritis this is speeded up. In the growth plate, long bones increase in length via increased proliferation of the chondrocytes but after proliferation occurs, the cells enter into the hypertrophic stage of differentiation. In fracture repair and bone induction in response to demineralized bone graft or bone morphogenetic protein (BMP), the transition occurs more quickly.
Most of the growth factors being examined for use in cartilage and bone were identified because they increased bone formation, either by increasing endochondral differentiation or by acting on osteoblasts directly. These growth factors have multiple effects. They promote differentiation of mesenchymal cells. For example, transforming growth factor .beta.(TGF.beta.) can induce mesenchymal cells to become cartilage cells in vitro or in vivo. BMP induces mesenchymal cells to become chondrocytes in vivo in mesenchymal tissues that would not normally support bone or cartilage formation. In bone, BMP causes the mesenchymal cells to become chondrocytes when oxygen tension is low or to become osteoblasts when oxygen tension is high.
Growth factors can also cause already-committed cells to differentiate further along their lineage. Factors like insulin-like growth factor (IGF's) or basic fibroblast growth factor (bFGF) do not affect differentiation of mesenchymal cells into cartilage or bone cells in vitro, but in vivo, they enhance the expression of a mature calcifying chondrocyte or osteoblast. TGF.beta.and BMPs also have effects on already-committed cells. All four of these factors cause resting zone chondrocytes to acquire a phenotype typical of hypertrophic cells. Thus, they will eventually calcify their matrix, supporting endochondral bone formation. If the goal is to enhance bone formation, this is good. But if the goal is to get stable, non-calcified cartilage, this outcome is exactly the opposite of what is desired.
It is an object of this invention, to provide a method to aid in the healing of cartilage wounds by enhancing chondrocyte production without causing further differentiation along the endochondral developmental pathway resulting in calcified cartilage.
Temporally, the release of PDGF from platelets is one of the initial events that occurs in the resolution of a wound. PDGF appears to enhance cartilage and bone formation (Lynch et al., 1994), but it has not previously been known whether this is due to an increase in the pool of less mature cells, or to a direct effect on the differentiation of those cells. PDGF is well known as a competence growth factor (Antoniades et al., 1982; Tsukamoto et al., 1991) and has been shown to initiate extensive proliferation of osteoblasts (Centrella et al., 1989; Hock et al., 1988).
Previous studies also have shown that PDGF regulates extracellular matrix synthesis by osteoblasts in addition to its effects on proliferation. The precise effect of PDGF on matrix synthesis is not clear, with some studies reporting an inhibition of collagen production (Canalis and Lian, 1988), and others reporting no change (Centrella et al. 1989).
Considerable similarities exist between the events that occur in endochondral bone development and those that occur during wound healing in bone. These processes involve the induction of mesenchymal cells into and along the chondrocyte lineage by chondrogenic growth factors like transforming growth factor beta (TGF.beta.) (Centrella et al., 1988; Crabb et al., 1990; Schwartz et al., 1993), insulin-like growth factor (IGF) (Demarquay et al., 1992; Sunic et al., 1995; Wroblewski et al., 1995), and basic fibroblast growth factor (bFGF) (Fujisato et al., 1996; Kato et al., 1990; Wang et al., 1993).
In bone wound healing in vivo, enhanced proliferation of osteochondroprogenitor cells is an important first step. Platelet-derived growth factor (PDGF), a cytokine which stimulates proliferation of mesenchymal cells in a broad range of tissues (Antoniades et al., 1982 ; Heldin et al., 1988; Ross et al., 1986; Stiles, 1983), is released from platelets at wound sites (Coughlin et al., 1980). In addition, PDGF is produced by osteoblasts (Zhang et al., 1991) and stored in bone (Hauschka et al., 1986), further increasing its local concentration, and as a result, increasing the pool of osteochondroprogenitor cells.
PDGF is a disulfide-linked dimer with a molecular weight of approximately 25 kDa (Coughlin et al., 1980). PDGF-BB is one of three isoforms of PDGF resulting from the dimeric combination of two distinct, but structurally related, polypeptide chains designated as A and B. Fibroblasts, smooth muscle cells, periodontal ligament cells, and osteoblastic cells have all been shown to respond to this cytokine (Centrella et al., 1989, Kinoshita et al., 1992; Pfeilschifter et al., 1990; Tsukamoto et al., 1991). In addition to its stimulatory effect on osteoblast proliferation (Centrelia et al., 1989; Canalis and Lian, 1988; Hock et al., 1994), other aspects of cell metabolism and phenotypic expression are affected as well.
Studies examining matrix production and differentiation markers have suggested that exposure of fetal rat calvarial cells to PDGF has an inhibitory effect on collagen synthesis and no effect on osteocalcin production (Canalis et al., 1998). Other studies by the same group on cells isolated from fetal rat parietal bone, while showing enhanced rates of increased collagenase-digestible and noncollagenase-digestible protein production, demonstrated no difference in the overall relative collagen synthesis (Centrella et al., 1989). These observations suggest that PDGF has an overall anabolic effect on the cells, but does not promote osteoblastic differentiation.
To date, attention to the effects of PDGF, particularly the BB isoform, on chondrocytes has been relatively limited. In vitro studies by Chen et al. (Chen et al., 1992) appear to indicate that chick limb bud mesodermal chondrogenesis is inhibited by PDGF. Others, however, have observed chondrogenic differentiation when cultures of perichondrial cells were stimulated with PDGF (Skoog et al., 1990).
It is also an object of this invention to teach how PDGF may be used to enhance proliferation, control matrix synthesis, and inhibit endochondral maturation of chondrocytes, cells whose regulation is essential to development of cartilage and endochondral bone.
Publications referred to herein are listed below. All publications referred to herein are hereby incorporated by reference.
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